Supplementary Methods
Patients
Newly diagnosed CLL patients from several Italian Institutions were prospectively enrolled within
12 months of diagnosis into the O-CLL1-GISL protocol (clinicaltrial.gov identifier:
NCT00917540). Recruitment began in January 2007 and the criteria for CLL diagnosis employed
followed the 1996 NCI/WG guidelines requiring >5,000 lymphocytes/µL in the peripheral blood
(1). CLL cell phenotype, CD38 and ZAP-70 expression, and IGHV mutational status were
performed in a central laboratory in Genova, while all FISH and genetic analyses were performed in
Milan. This study was approved by the ethics committees from each participating center. All
patients provided written informed consent to participate in the study. To date the study has
enrolled 480 Binet stage A patients.
Assessment of biological markers
Cytogenetic abnormalities involving deletions at chromosomes 11q23, 13q14, 17p13, and trisomy
12 were evaluated by FISH on a purified CD19+ population (2). CD38-positive leukemic cells were
measured by triple staining with CD19-FITC, CD38-PE (both BD Biosciences), and CD5-PC5
(Beckman Coulter). The cells were analyzed using a FACS Calibur flow-cytometer (BD
Biosciences). CD38-positive cases were indicated as those having greater than 30% positive cells
(3). ZAP-70 was determined by flow-cytometry with a ZAP-70-FITC (clone 2F3.2, Millipore) or an
isotype control mAb (mouse IgG2a-FITC, BD Biosciences) as previously described (4,5). We
previously demonstrated that 40% of ZAP-70-positive cells represents the best cut-off value for
predicting TFS (6), thus, this threshold was used to identify ZAP-70-positive cases in the O-CLL1GISL cohort. IGHV mutational status was assessed using cDNA (5). Sequences were aligned to the
IMGT directory and analyzed using IMGT/VQUEST software.
Indications for therapy
All patients underwent follow-up visits every 6 months. All physicians used the NCI/WG
guidelines as reference criterion for starting therapy (1,7) and all patients had a minimum follow-up
of 6 months and were evaluated for TFS.
Statistical analysis
For categorical variables, statistical comparisons were performed using two-way tables for the
Fisher’s exact test and multi-way tables for the Pearson’s Chi-square test. TFS was calculated from
diagnosis to the first CLL treatment (event) or to last follow-up (censoring). TFS analyses were
performed using the Kaplan-Meier method. Statistical significance of associations between
individual variables and TFS was calculated using the log-rank test. Independent correlates of the
outcome variable were identified by univariate and multiple Cox regression analyses. A value of
P<0.05 was considered statistically significant. Continuous variables of prognostic importance on
TFS in univariate proportional hazards Cox regression were dichotomized using published
thresholds and laboratory norms. ROC curves identified additional thresholds. Robustness (i.e. the
internal validity) of the multivariate Cox model was assessed by a bootstrapping resampling
technique. In the bootstrapping method, the same multivariate Cox model was fitted 1,000 times
using bootstrap sample. Harrell C-statistics were calculated to further evaluate discriminatory value
of the progression-risk score (c=1 indicates perfect discrimination; c=0.5 indicates complete
absence of prognostic accuracy). The prognostic accuracy of the risk score was further investigated
by the Hosmer-May test (a test for analyzing calibration in Cox models) (8), explained variation on
outcome (an index combining calibration and discrimination) (9), and Akaike information criterion
(AIC) (10). The lower the AIC, the higher the prognostic accuracy of a predictive model. The
chances of a given prognostic model to be the best model (among the two candidate models) were
assessed by calculating Akaike weights. To calculate these weights, firstly we calculate for each
model the relative likelihood, which is just the exp (-0.5 *∆AIC score for that model). The Akaike
weight for the model under investigation is this figure divided by the sum of these values across all
candidate models. The Harrell C-statistic was performed using STATA version 9; all the other
analyses were performed using SPSS Statistics 21.
Comparison of progression-risk score with the MD Anderson model
Wierda et al proposed a prognostic model for predicting TFS in CLL (12). To compare our
progression-risk score with this model, we calculated the total point score in our cohort using the
formula proposed by Wierda:
I(No. of lymph node sites involved=3) × 7.370 + I(FISH=del11q) × 9.312 + I(FISH=del17p)
× 11.285 + (diameter of largest cervical lymph node in cm) × 4.172 + (LDH/100) × I([IGHV
gene=mutated] × 5.000 + (LDH÷100) × I(IGHV gene=unmutated)×1.065] + 35.467.
The indicator function (I) is equal to 1 if the statement in the parentheses is true and is equal to 0
otherwise.
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